Branched chain fatty acids for prevention or treatment of gastrointestinal disorders

ABSTRACT

The present invention is directed to a method of preventing or treating a gastrointestinal condition in a subject, that includes administering one or more branched chain fatty acid to the subject under conditions effective to prevent 5 or treat the gastrointestinal condition in the subject. The present invention is also directed to methods of promoting gastrointestinal health in a subject and propagation of probiotic organisms. Also disclosed is a formulation which includes one or more branched chain fatty acid and an aqueous phase emulsified with the one or more branched chain fatty acids, where the formulation includes 10 over 25 wt % of the one or more branched chain fatty acid.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/972,992, filed Sep. 17, 2007, which is herebyincorporated by reference in its entirety.

The subject matter of this application was made with support from theUnited States Government under National Institutes of Health (NIH) GrantNo. GM071534. The U.S. government has certain rights.

FIELD OF THE INVENTION

The present invention is directed to methods for prevention or treatmentof gastrointestinal disorders.

BACKGROUND OF THE INVENTION

Branched chain fatty acids (BCFA) are mostly saturated fatty acids (SFA)with one or more methyl branches on the carbon chain. BCFA aresynthesized mainly by the skin and have long been known to be a majorcomponent of vernix caseosa (10-20% dry weight) (Nicolaides et al, “SkinLipids. 3. Fatty Chains in Skin Lipids. The Use of Vernix Caseosa toDifferentiate between Endogenous and Exogenous Components in Human SkinSurface Lipid,” J Am Oil Chem Soc 42:702-707 (1965)). Among terrestrialanimals, vernix is unique to humans, and is not found in other landmammals, including other primates (Pickens et al., “Characterization ofVernix Caseosa: Water Content, Morphology, and Elemental Analysis,” JInvest Dermatol 115:875-881 (2000)). Vernix is made of sebum and fetalcorneocytes (Nicolaides et al., “Skin Lipids. 3. Fatty Chains in SkinLipids. The Use of Vernix Caseosa to Differentiate between Endogenousand Exogenous Components in Human Skin Surface Lipid,” J Am Oil Chem Soc42:702-707 (1965) and Narendran et al., “Interaction between PulmonarySurfactant and Vernix: A Potential Mechanism for Induction of AmnioticFluid Turbidity,” Pediatr Res 48:120-124 (2000)) and is produced byfetal skin starting at 24 weeks gestational age and continuing untilterm birth (Moore et al., “Fetal Cocaine Exposure: Analysis of VernixCaseosa,” J Anal Toxicol 20:509-511 (1996)). During the third trimestervernix sloughs off as particulates that become suspended in amnioticfluid (Narendran et al., “Interaction between Pulmonary Surfactant andVernix: A Potential Mechanism for Induction of Amniotic FluidTurbidity,” Pediatr Res 48:120-124 (2000) and Yoshio et al.,“Antimicrobial Polypeptides of Human Vernix Caseosa and Amniotic Fluid:Implications for Newborn Innate Defense,” Pediatr Res 53:211-216(2003)), possibly aided by lung surfactant phospholipids that also enterthe amniotic fluid. The fetus normally swallows amniotic fluid inamounts approaching 500 ml at the end of gestation (Miettinen et al.,“Gas-liquid Chromatographic and Mass Spectrometric Studies on Sterols inVernix Caseosa, Amniotic Fluid and Meconium,” Acta Chem Scand22:2603-2612 (1968) and Sherman et al., “Fetal Swallowing: Correlationof Electromyography and Esophageal Fluid Flow,” Am J Physiol258:R1386-1394 (1990)) and with it vernix. Thus, the late term fetal gutis normally exposed to vernix and its BCFA, increasingly so asparturition approaches.

Vernix dry matter is composed of approximately equal amounts of proteinand lipids (Pickens et al., “Characterization of Vernix Caseosa: WaterContent, Morphology, and Elemental Analysis,” J Invest Dermatol115:875-881 (2000) and Hoeger et al., “Epidermal Barrier Lipids in HumanVernix Caseosa: Corresponding Ceramide Pattern in Vernix and FetalSkin,” Br J Dermatol 146:194-201 (2002)). Lipid fractions in vernix havebeen comprehensively characterized (Nicolaides et al., “The Fatty Acidsof Wax Esters and Sterol Esters from Vernix Caseosa and from Human SkinSurface Lipid,” Lipids 7:506-517 (1972); Rissmann et al., “New Insightsinto Ultrastructure, Lipid Composition and Organization of VernixCaseosa,” J Invest Dermatol 126:1823-1833 (2006) and Kaerkkaeinen etal., “Lipids of Vernix Caseosa,” J Invest Dermatol 44:333-338 (1965))and shown to be 25-30% sterol esters (SE), 18-36% triglycerides (TAG),12-16% wax esters (WE), 9% squalene, 5% ceramides, and low levels ofnon-esterified fatty acid (NEFA) fraction was also detected by some(Rissmann et al., “New Insights into Ultrastructure, Lipid Compositionand Organization of Vernix Caseosa,” J Invest Dermatol 126:1823-1833(2006) and Tollin et al., “Vernix Caseosa as a Multi-component DefenceSystem Based on Polypeptides, Lipids and Their Interactions,” Cell MolLife Sci 62:2390-2399 (2005)) but not by others (Nazzaro-Porro et al.,“Effects of Aging on Fatty Acids in Skin Surface Lipids,” J InvestDermatol 73:112-117 (1979)). BCFA are found in all acyl-carrying lipidclasses, WE (16-53%) and SE (27-62%) (Nicolaides et al., “The FattyAcids of Wax Esters and Sterol Esters from Vernix Caseosa and from HumanSkin Surface Lipid,” Lipids 7:506-517 (1972); Rissmann et al., “NewInsights into Ultrastructure, Lipid Composition and Organization ofVernix Caseosa,” J Invest Dermatol 126:1823-1833 (2006); Kaerkkaeinen etal., “Lipids of Vernix Caseosa,” J Invest Dermatol 44:333-338 (1965) andNazzaro-Porro et al., “Effects of Aging on Fatty Acids in Skin SurfaceLipids,” J Invest Dermatol 73:112-117 (1979)), as well as in the TAG(18-21%) and NEFA (21%) fractions (Rissmann et al., “New Insights intoUltrastructure, Lipid Composition and Organization of Vernix Caseosa,” JInvest Dermatol 126:1823-1833 (2006)).

Apart from skin (Nicolaides et al., “Skin Lipids. 3. Fatty Chains inSkin Lipids. The Use of Vernix Caseosa to Differentiate betweenEndogenous and Exogenous Components in Human Skin Surface Lipid,” J AmOil Chem Soc 42:702-707 (1965); Nicolaides et al., “The Fatty Acids ofWax Esters and Sterol Esters from Vernix Caseosa and from Human SkinSurface Lipid,” Lipids 7:506-517 (1972) and Nicolaides et al., “SkinLipids: Their Biochemical Uniqueness,” Science 186:19-26 (1974)), BCFAare at very low levels in internal tissue (Nicolaides et al., “SkinLipids: Their Biochemical Uniqueness,” Science 186:19-26 (1974)), butare also found in human milk (Jensen et al., “Handbook of MilkComposition,” Academic Press Inc., San Diego, (1995); Egge et al.,“Minor Constituents of Human Milk. IV. Analysis of the Branched ChainFatty Acids,” Chem Phys Lipids 8:42-55 (1972) and Gibson et al., “FattyAcid Composition of Human Colostrum and Mature Breast Milk,” Am J ClinNutr 34:252-257 (1981)) at concentrations as high as 1.5% w/w of totalfatty acids (FA). This level is comparable to and in some cases greaterthan that of docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid(ARA, 20:4n-6) in the same milk. For instance, a 1981 publicationreported the concentration of anteiso 17:0 in Australian women'scolostrum to be 0.45% w/w of total FA, exceeding the concentrations ofDHA (0.32% w/w) and ARA (0.4% w/w) (Gibson et al., “Fatty AcidComposition of Human Colostrum and Mature Breast Milk,” Am J Clin Nutr34:252-257 (1981)).

Meconium, the newborn's first fecal pass, first appears in the fetal GItract at around 12 weeks of gestational age, and is normally passedafter birth (Ahanya et al., “Meconium Passage In Utero: Mechanisms,Consequences, and Management,” Obstet Gynecol Surv 60:45-56 (2005);Gareri et al., “Drugs of Abuse Testing in Meconium,” Clin Chim Acta366:101-111 (2006); and Ostrea et al., “Fatty Acid Ethyl Esters inMeconium: Are They Biomarkers of Fetal Alcohol Exposure and Effect?”Alcohol Clin Exp Res 30:1152-1159 (2006)). It consists of amniotic fluidresidue, skin and gastrointestinal (GI) epithelial cells, GI secretionsand enzymes, lipids, sugars, proteins, cholesterol, sterols, bile acidand salts (Ahanya et al., “Meconium Passage In Utero: Mechanisms,Consequences, and Management,” Obstet Gynecol Surv 60:45-56 (2005);Gareri et al., “Drugs of Abuse Testing in Meconium,” Clin Chim Acta366:101-111 (2006); Buchanan et al., “Chemical Comparison of NormalMeconium and Meconium from a Patient with Meconium Ileus,” Pediatrics9:304-310 (1952); and Righetti et al., “Proton Nuclear MagneticResonance Analysis of Meconium Composition in Newborns,” J PediatrGastroenterol Nutr 36:498-501 (2003)). Meconium contains 12% dry weightlipid (Buchanan et al., “Chemical Comparison of Normal Meconium andMeconium from a Patient with Meconium Ileus,” Pediatrics 9:304-310(1952)), and there is only one unconfirmed study reporting BCFA inmeconium (Terasaka et al., “Free Fatty Acids of Human Meconium,” BiolNeonate 50:16-20 (1986)). There are no studies linking BCFA compositionof vernix and meconium in the same infants.

It was hypothesized that vernix BCFA of term newborns would survive thealimentary canal and be found in meconium. The test of this hypothesisled to characterizing the relative BCFA profiles of vernix and meconiumto establish the degree to which the profile is altered by the sterilefetal gut in utero.

The present invention is directed to overcoming the deficiencies in theart.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method ofpreventing or treating a gastrointestinal condition in a subject thatincludes administering one or more branched chain fatty acid to thesubject under conditions effective to prevent or treat thegastrointestinal condition in the subject.

A second aspect of the present invention relates to a method ofpromoting gastrointestinal health in a subject that includesadministering one or more branched chain fatty acid to the subject underconditions effective to promote gastrointestinal health in the subject.

A third aspect of the present invention relates to a method of promotingpropagation of probiotic organisms that includes providing a populationof cells comprising probiotic organisms and administering one or morebranched chain fatty acid to the population of cells under conditionseffective to promote propagation of probiotic organisms in thepopulation of cells.

A fourth aspect of the present invention relates to a formulation whichincludes one or more branched chain fatty acid and an aqueous phaseemulsified with the one or more branched chain fatty acid, where theformulation includes over 25 wt % of the one or more branched chainfatty acid.

Vernix suspended in amniotic fluid is normally swallowed by the lateterm fetus. It was hypothesized that branched chain fatty acids (BCFA),long known to be major vernix components, would be found in meconium andthat the profiles would differ systematically. Vernix and meconium werecollected from term newborns and analyzed. BCFA-containing lipidsconstituted about 12% of vernix dry weight, and were predominantlysaturated, and had 11 to 26 carbons per BCFA. In contrast, meconium BCFAhad 16 to 26 carbons, and were about 1% of dry weight. Meconium BCFAwere mostly in the iso configuration, whereas vernix BCFA containeddimethyl and middle chain branching, and five anteiso BCFA. The mass ofBCFA entering the fetal gut as swallowed vernix particles is estimatedto be 180 mg in the last month of gestation while the total mass of BCFAfound in meconium is estimated to be 16 mg, thus most BCFA disappearfrom the fetal gut. The BCFA profiles of vernix and meconium show thatBCFA are major components of normal healthy term newborngastrointestinal tract. BCFA are candidates for agents that play a rolein gut colonization and should be considered a nutritional component forthe fetus/newborn.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows BCFA methyl esters profiles for vernix and meconium(means±SEM, n≦18 newborn) listed left to right in order of molecularweight. Means are for those FA appearing in samples from at least threenewborns. iso-BCFA have a dimethyl terminal structure: iso-16:0 issynonymous with 14-methyl-15:0 (14-methyl-pentadecanoic acid).anteiso-BCFA have a methyl branch at the n-2 position: anteiso-17:0 issynonymous with 14-methyl-16:0 (14-methyl-hexadecanoic acid). i-iso;ai=anteiso; Me=methyl; diMe=two methyl branches. Key: □ vernix; ▪meconium. *p<0.05

FIG. 2 shows Iso BCFA methyl esters in vernix and meconium (means±SEM,n≦18 newborn) listed left to right in order of molecular weight. i=iso.Key: □ vernix; ▪ meconium. *p<0.05

FIG. 3 shows Anteiso BCFA methyl esters in vernix and meconium(means±SEM, n≦18 newborn) listed left to right in order of molecularweight. ai-anteiso. Key: □ vernix; ▪ meconium. *p<0.05

FIG. 4 shows normal (straight chain) FAME in vernix (means±SEM, n≦18newborn) listed left to right in order of molecular weight. Key: □vernix; ▪ meconium. *p<0.05

FIG. 5A-C shows GC/MS reconstructed ion chromatograms (RIC) from Caco-2BCFA uptake experiment. RIC section shows saturated and monounsaturatedC14-18 fatty acids. FIGS. 5A and 5C are scaled to match for n-16:0 andthe 18:1 isomers for convenient visual comparison of relativeabundances. FIG. 5A shows control Caco-2 cells showing no significantBCFA. FIG. 5B shows a mixture of pure BCFA (iso-14:0, anteiso-17;0,iso-18:0, iso-20:0). FIG. 5C shows total FA from BCFA-treated BCFA inFIG. 5B. iso-16:0 is detected as a novel product bio-synthesized by thecells, most likely to be derived from iso-14:0 from chain elongation.RIC are reconstructed from full mass spectra collected each one second,positively identifying the structural assignments.

FIG. 6A-E shows phospholipids fatty acids purified from Caco-2 cellstreated with BCFA. FIG. 6A shows RIC of native (untreated) cells. FIG.6B shows RIC of BCFA treated cells. FIG. 6C shows a selected ionchromatogram showing elution of 16:0's. FIG. 6D shows the mass spectrumof the first peak in FIG. 6B, characteristic of newly synthesizediso-16:0. FIG. 6E shows the mass spectrum of n-16:0, normally present incells, with negligible m/z 255 peak (loss of methyl).

FIG. 7 shows monoacylglycerol (MAG) FA from BCFA-treated Caco-2 cells.The BCFA mixture dominates MAG FA, and they are represented in closeproportion to the parent free fatty acid mixture shown in FIG. 5B.Compare iso-20:0 to iso-14:0 peak heights to those of FIG. 6B. Note alsothat the iso-16:0 to iso-14:0 ratio is small compared to that of FIG.6B, indicating relatively low biosynthesis of iso-16:0 appearing in thislipid class.

FIG. 8 shows BCFA profiles in a parent free fatty acid mixture and inlipids of BCFA-treated Caco-2 cells. Iso-16:0 is present in all cellextracts. Note especially the selection against iso-20:0 in PL, andfavoring iso-20:0 in MAG.

FIG. 9 shows B. breve (TSB) or S. inulinus (TSB or NB) growth isenhanced with vernix compared to a control. Pathogen growth was notaffected by vernix. The higher offset for the vernix curves is due toundissolved particulates in vernix.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a method of preventing ortreating a gastrointestinal condition in a subject that includesadministering one or more branched chain fatty acid to the subject underconditions effective to prevent or treat the gastrointestinal conditionin the subject.

A second aspect of the present invention relates to a method ofpromoting gastrointestinal health in a subject that includesadministering one or more branched chain fatty acid to the subject underconditions effective to promote gastrointestinal health in the subject.

For each method, a subject in need may be selected. The method ofpreventing or treating a gastrointestinal condition in a subject may becarried out in a human, in particular, in a fetus, infant, newborninfant, child, or an adult.

The gastrointestinal condition can be mediated by infection of thesubject's gastrointestinal tract by a pathogenic bacteria (e.g.,necrotizing enterocolitis) or may require microbial colonization of thesubject's gastrointestinal tract (e.g., in conjunction with anantibiotic treatment). The gastrointestinal condition may also be adisease of the intestine involving inflammation, such as inflammatorybowel disease.

In general, branched chain fatty acids, in accordance with the presentinvention, may be non-esterified fatty acids or covalently linked to alipid, including wax esters, sterol esters, triacylglycerols, or anyother lipid-related molecular species, natural or artificial.

The branched chain fatty acid can be a C₁₁ to C₂₆ branched chain fattyacid and mixtures thereof. The branched chain fatty acid may be4,7-dimethyl-nonanoic acid, 4,8-dimethyl-decanoic acid,8-methyl-undecanoic acid, iso-dodecanoic acid, 4,8-dimethyl-undecanoicacid, 4,9-dimethyl-undecanoic acid, iso-tridecanoic acid,anteiso-tridecanoic acid, 4,10-dimethyl-dodecanoic acid,iso-tetradecanoic acid, 4,11-dimethyl-tridecanoic acid,iso-pentadecanoic acid, anteiso-pentadecanoic acid,8,10-dimethyl-tetradecanoic acid, 4,12-dimethyl-tetradecanoic acid,iso-hexadecanoic acid, 2-methyl hexadecanoic acid,4,11-dimethyl-pentadecanoic acid, 4,13-dimethyl-pentadecanoic acid,iso-heptadecanoic acid, anteiso heptadecanoic acid, iso-octadecanoicacid, iso-eicosanoic acid, anteiso-heneicosanoic acid, iso-dodecanoicacid, iso-tetracosanoic acid, iso-pentacosanoic acid,anteiso-pentacosanoic acid, iso-hexacosanoic acid, phytanic acid,pristanic acid, or mixtures thereof.

The branched chain fatty acid may also be saturated and monounsaturatedfatty acids or mixtures thereof.

Further, the branched chain fatty acid can be a branched form of a fattyacid such as an octanoic acid, a decanoic acid, a lauric acid, amyristic acid, a palmitic acid, a stearic acid, an eicosanoic acid, apalmitoliec acid, an oleic acid, or mixtures thereof.

The therapeutic agent, regardless of its mode of action, can beadministered to a patient in the form of a pharmaceutical compositionthat also includes a pharmaceutically-acceptable carrier. Thepharmaceutical composition may be in a liquid or solid dosage formincluding, but not limited to, tablets, capsules, powders, solutions,suspensions, or emulsions.

The therapeutic agent of the present invention, and thus thepharmaceutical compositions of the present invention, can beadministered orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, byimplantation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, transdermally, byapplication to mucous membranes (such as, that of the nose, throat, andbronchial tubes), or by introduction into one or more lymph nodes.

In addition to being used in pharmaceutical compositions, branched chainfatty acids, in accordance with the present invention, can be added toany of a variety of food products, including milk, infant formula, babyfood, dietary supplements, vegetable oils, mayonnaise and condiments,yogurt, margarine and spreads, shortenings, and any food that includesfat or can be formulated with fat.

A third aspect of the present invention relates to a method of promotingpropagation of probiotic organisms that includes providing a populationof cells comprising probiotic organisms and administering one or morebranched chain fatty acid to the population of cells under conditionseffective to promote propagation of probiotic organisms in thepopulation of cells.

The method of promoting propagation of probiotic organisms may becarried out in vitro or in vivo.

The probiotic organism may be selected from the group consisting ofLactobacillus species, Bifidobacterium species, other lactic acidbacteria, and nonlactic acid bacteria, and mixtures thereof.

The probiotic organism may be a Lactobacillus species, such as L.acidophilus, L. amylovorus, L. brevis, L. casei, L. casei subsp.rhamnosus (Lactobacillus GG), L. caucasicus, L. crispatus, L.delhrueckii subsp. bulgaricus (L. bulgaricus), L. fermentum (L.fermenti), L. gasseri, L. helveticus, L. johnsonii, L. lactis, L.leichmannii, L, paracasei, L. plantarum, L. reuteri, L, rhamnosus ormixtures thereof.

The probiotic organism may be a Bifidobacterium species, such as B.adolescentis, B. bifidum, B. breve, B. infantis, B. lactis (B.animalis), B. licheniformis, B. longum or mixtures thereof.

The probiotic organism may be a lactic acid bacteria, such asEnterococcus faecium, Lactococcus lactis, Leuconstoc mesenteroides,Pediococcus acidilactici, Streptococcus thermophilus, or mixturesthereof.

The probiotic organism may be a nonlactic acid bacteria, such asBacillus subtilis, Escherichia coli strain nissle, Saccharomycesboulardii, Saccharomyces cerevisiae or mixtures thereof.

To the extent, the method of propagating probiotic organisms is carriedout in subjects, it is formulated and administered in substantially thesame manner as noted above.

Probiotics have proven effective in NEC (necrotizing enterocolitis)trials, specifically combinations of Lactobacillus acidophilus andBifidobacterium infantis (Hoyos A B., “Reduced Incidence of NecrotizingEnterocolitis Associated with Enteral Administration of Lactobacillusacidophilus and Bifidobacterium infantis to Neonates in an IntensiveCare Unit,” Int J Infect Dis 3:197-202 (1999) and Lin et al., “OralProbiotics Reduce the Incidence and Severity of NecrotizingEnterocolitis in Very Low Birth Weight Infants,” Pediatrics 115:1-4(2005), which are hereby incorporated by reference in their entirety) orBifidobacteria infantis, Streptococcus thermophilus, and Bifidobacteriabifidus (Bin-Nun et al., “Oral Probiotics Prevent NecrotizingEnterocolitis in Very Low Birth Weight Neonates,” J Pediatr 147:192-6(2005), which is hereby incorporated by reference in its entirety).

Probiotic bacteria are sold mainly in fermented foods and dairyproducts, specifically, yogurt-like products form the largest segment ofthe market for probiotic products (Heller et al., “Probiotic Bacteria inFermented Foods: Product Characteristics and Starter Organisms,” Am JClin Nutr 73(suppl):374S-9S (2001), which is hereby incorporated byreference in its entirety). As used herein, “probiotics” are defined asviable microorganisms, sufficient amounts of which reach the intestinein an active state and thus exert positive health effects (de Vrese etal., “Probiotics, Prebiotics, and Synbiotics,” Adv Biochem EngBiotechnol 111:1-66 (2008), which is hereby incorporated by reference inits entirety).

A fourth aspect of the present invention relates to a formulation whichincludes one or more branched chain fatty acid and an aqueous phaseemulsified with the one or more branched chain fatty acid, where theformulation includes over 25 wt % of the one or more branched chainfatty acid.

The formulation may include up to 98 wt %, preferably up to 70 wt %, ofthe branched chain fatty acid or a mixture thereof. In addition, thebranched chain fatty acid or a mixture thereof of the formulation mayinclude up to 20 wt % of isomyristic acid, isopentadecoanoic acid,anteisopentadecanoic acid, isopalmitic acid, isoeicosanoic acid,anteisoeicosanoic acid, isodocosanoic acid, isotetracosanoic acid,isohexacosanoic acid or combinations thereof. The formulation may alsoinclude an emulsifier.

Vernix has 29% BCFA, meconium is about 12%, and breastmilk is about 1%.Vernix and meconium are present in the fetus and the immediate newborn,while breastmilk applies after birth. The formulation of the presentinvention is designed to achieve enhanced results compared to vernix. Itis advantageous for the BCFA to be emulsified in a water (aqueous)phase. Such an emulsion does not correspond to anything found in vernixor meconium, or breastmilk. A suitable BCFA formulation for preventionor treatment of abnormal bacterial flora in the gastrointestinal (GI)tract, and for optimization of intestinal health, would be a mixture offat and aqueous solution of up to 50% fat. The aqueous phase includesany solution compatible with the GI tract. The mixture should beemulsified with any food grade emulsifier acceptable for pediatric use,such as soy lecithin.

Examples

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Materials and Methods for Examples 1-5 Sample Collection

Eighteen samples of vernix and meconium were collected from 18 normalterm newborns at Cayuga Medical Center in Ithaca, N.Y. Vernix wasremoved from the shoulder regions in the birthing room, placed in cleantubes and stored at −80° C. until analysis. Meconium was collected fromdiapers and similarly transferred into clean tubes and stored at −80° C.until analysis.

FA Analysis

Total lipids were extracted from the vernix and the meconium samplesaccording to a modified Bligh and Dyer method (Bligh et al., “A RapidMethod of Total Lipid Extraction and Purification,” Can J BiochemPhysiol 37:911-917 (1959), which is hereby incorporated by reference inits entirety). Fatty acids are overwhelmingly found in mammalian pools,such as vernix and meconium, as acyl moieties which are constituents ofhigher molecular weight lipid molecules such as TAG, SE, and WE. Fordetailed molecular analysis, fatty acyl groups are hydrolyzed and fattyacid methyl esters (FAME) synthesized for analysis. FAME were preparedusing 14% BF3 in methanol (Sigma Chemical, St. Louis, Mo.). Butylatedhydroxytoluene (BHT) was added to methanol as an antioxidant.Heptadecanoic acid (Sigma Chemical, St Louis, Mo.) in chloroform wasused as an internal standard. This routine step obscures heptadecanoicacid which is normally rare in mammalian tissue but is present in vernixand meconium. Because of the extraordinary diversity of FA in thesesamples, any internal standard interferes with analysis of one or moreFA in some of the samples. A correction was applied to estimate theextent of interference, and the signals were carefully calibratedagainst external standards.

FAME analyses were performed using a Hewlett Packard 5890 series H gaschromatograph (GC). A BPX-70 column (60 m×0.32 mm×0.25 μm, SGE, Austin,Tex.) was used for the analysis with H₂ as the carrier gas. FAMEidentities were determined by a chemical ionization (CI) and electronimpact (EI) mass spectrometry (MS), using a Varian Star 3400 GC coupledto a Varian Saturn 2000 ion trap MS. BCFA FAME identities were based onGC retention time of each substance and its electron impact massspectra. FAME mass spectral assignments were confirmed by conversion ofthe FAME to picolinyl ester derivatives according to the methoddescribed by Yang et al. (Yang et al., “Picolinyl Ester FragmentationMechanism Studies with Application to the Identification ofAcylcarnitine Acyl Groups Following Transesterification,” J Am Soc MassSpectrom 17:1620-1628 (2006), which is hereby incorporated by referencein its entirety), followed by GC/MS analysis and comparison toliterature spectra (Carballeira et al., “Characterization of NovelMethyl-branched Chain Fatty Acids from a Halophilic Bacillus Species,” JNat Prod 64:256-259 (2001); Suutari et al., “Signature GLC-MS Ions inIdentification of Delta 5 and Delta 9 Unsaturated Iso and AnteisoBranched Chain Fatty Acids,” J Microbiol Methods 17:39-48 (1993);Karlsson et al., “Studies on Feather Waxes of Birds,” Arkiv for Kemi31:143-158 (1969); Yu et al., “Location of Methyl Branchings in FattyAcids: Fatty Acids in Uropygial Secretion of Shanghai Duck by GC-MS of4,4-Dimethyloxazoline Derivatives,” Lipids 23:804-810 (1988); Apon etal., “The Determination of the Position Isomers of the Methyl BranchesFatty Acids Methyl Esters by Capillary GC/MS,” J Chromatogr Sci13:467-473 (1975); and Harvey et al., “Identification of Long-chainFatty Acids and Alcohols from Human Cerumen by the Use of Picolinyl andNicotinate Esters,” Biomed Environ Mass Spectrom 18:719-723 {1989),which are hereby incorporated by reference in their entirety).

An equal weight FAME mixture (68A; Nuchek Prep, Elysian, Minn.) was usedto calculate response factors. The following were also used asstandards: n-11:0 up to n-24:0 (Nuchek Prep, Elysian, Minn.); iso 13:0,anteiso 13:0, iso 15:0, anteiso 15:0; iso 17:0, anteiso 17:0 (LarodanFine Chemicals AB, Malmo, Sweden) and 10 methyl hexadecanoic acid(Matreya LLC, Pleasant Gap, Pa.). FA levels were expressed as weight %of total fatty acids for all 11 to 32 carbons FA.

Statistics

Data are expressed as mean±SD for study population characteristics, andas mean±SEM for FA analysis. Statistical analyses were made using JMP 6(SAS Institute, Cary, N.C.). Differences in mean of each FA werecalculated using one sample t-test for non-zero differences, with p<0.05declared significant.

Subjects

Characteristics of the study population are presented in Table 1. Nocomplications were present for any of the newborns other than as noted.All but two newborns were by vaginal delivery. Six mothers receivedantibiotic treatment during pregnancy; five of them gave birth to femaleinfants.

TABLE 1 Characteristics of study population (mean ± SD (range)).Mother's age (years)  29 ± 5.8 18-42 Gestational age (weeks) 40 ± 1 38-41 Birthweight (kg) 3.3 ± 0.5 2.3-4.4 Gender 10 female, 8 malesDelivery 16 vaginal, 2 CS* Antibiotics 5 female, 1 male *Cesariansection

Example 1 Overall FA Distribution

A profile of FA classes is shown in Table 2. Comparisons of all classeswere significant at the p<0.05 level. BCFA constituted almost a third(29.1±1.5% w/w) of all FA in vernix and were significantly highercompared to the mean levels in meconium (17.5±1.3% w/w; p<0.05). Thisdrop in BCFA was accompanied by a reciprocal increase in normal (n-)saturated FA (n-SFA) specifically, 34±1.9% w/w in vernix and 51.3±3.0%w/w in meconium (p<0.05). Differences in n-monounsaturated fatty acids(MUFA) and polyunsaturated fatty acid (PUFA) were modest by comparison.

TABLE 2 Profile of FA classes (% w/w) in vernix and meconium (mean ±SEM) FA Vernix Meconium BCFA 29.1 ± 1.5* 17.5 ± 1.3 n-SFA   34 ± 1.9*51.3 ± 3.0 n-MUFA 31.0 ± 1.7* 22.4 ± 2.1 PUFA  3.9 ± 0.4*  7.1 ± 1.1 *p< 0.05

Overall, BCFA hydrolyzed from their native lipid classes constituted5.8% of dry weight of vernix, corresponding to approximately 12% of dryweight of vernix within the native BCFA-containing lipids. Meconium had0.55% dry weight of hydrolyzed BCFA and an estimated 1% ofBCFA-containing lipids.

Example 2 BCFA Distribution in Vernix and Meconium

FIG. 1 is a graphical summary of the BCFA profiles for vernix andmeconium for those BCFA detected in samples from at least 3 newborns,presented left to right in order of carbon number. In total, 30 BCFAwere identified in vernix while nine were also detected in meconium.Vernix BCFA ranged from 11 to 26 carbon atoms and were primarilysaturated apart from two iso monounsaturates. iso BCFA, anteiso BCFA,middle chain monomethyl BCFA and dimethyl BCFA were all detected amongvernix BCFA. In contrast, meconium BCFA had a much more restricted rangeof carbon numbers, from 16 to 26 carbons. Of the nine meconium BCFA,eight were iso BCFA, of which two were MUFA, and one was anteiso.

Example 3 Profile of the iso BCFA in Vernix and Meconium

As shown in FIG. 2, the vernix iso BCFA profile had odd and even carbonnumbered FA from iso-12:0 to iso-16:0, and only even carbon numbers atgreater chain lengths. In contrast, meconium iso BCFA was dominated bythe shortest chain BCFA in its profile, iso-16:0, which was more thantwice the relative concentration of any other BCFA. Five of the eightiso-BCFA appearing in both vernix and meconium were a significantlydifferent proportion of BCFA in the respective profiles; thepreponderance of longer chains in meconium lead to significantdifferences in three of the four iso-BCFA of chain numbers from 20 to 26carbons.

Example 4 Profile of anteiso BCFA

As shown in FIG. 3, all five anteiso BCFA detected in vernix are oddcarbon numbered. They range from 13 to 25 carbons, and only one,anteiso-17:0, is found in meconium.

Example 5 Profile of the Straight Chain, n-FA Profiles for Vernix andMeconium

As shown in FIG. 4, vernix normal FA had 11 to 26 carbon atoms, andmeconium FA had 14 to 26 carbons, and both contained small amounts ofodd chain number FA. Again, meconium BCFA tended to be of greatermolecular weight.

The presence of BCFA in both vernix and meconium of healthy term infantsindicates that BCFA are a major component of gut contents of normal termnewborns, and their presence in meconium implies that they are presentthroughout the length of the gut. As such, BCFA are a component of theGI tract milieu present when the first few environmental microorganismsappear in the initial stage of gut colonization during and immediatelyafter parturition or Cesarean section. In meconium, the systematic shiftin BCFA profiles to high molecular weights, as well as the absence ofmost BCFA other than iso-BCFA, indicates that the fetal alimentary canalreadily absorbs and metabolizes most BCFA.

There are many reports of BCFA in breastmilk, with the earliest and mostextensive, showing 54 BCFA with a cumulative concentration of 1.5% w/w(Egge et al., “Minor Constituents of Human Milk. IV. Analysis of theBranched Chain Fatty Acids,” Chem Phys Lipids 8:42-55 (1972), which ishereby incorporated by reference in its entirety). A 1981 paper measuredthe concentration of BCFA in mature Australian breast milk to be a totalof 0.84% w/w and one BCFA, anteiso-17:0 in colostrum at 0.45% w/w offatty acids, exceeding the concentrations of DHA (0.32% w/w) and ARA(0.40% w/w) in the same mother's mature breastmilk (Gibson et al.,“Fatty Acid Composition of Human Colostrum and Mature Breast Milk,” Am JClin Nutr 34:252-257 (1981), which is hereby incorporated by referencein its entirety). Chen reported four BCFA with a cumulativeconcentration of 0.58%, w/w in Canadian breastmilk (Chen et al., “TransFatty Acid Isomers in Canadian Human Milk,” Lipids 30:15-21 (1995),which is hereby incorporated by reference in its entirety. A study ofCalifornia women yielded an average of 0.60% BCFA for the four BCFA thatwere reported (15:0, 16:0, 17:0, 18:0, branch location not reported)(Aitchison et al., “Influence of Diet on Trans Fatty Acids in HumanMilk,” Am J Clin Nutr 30:2006-2015 (1977), which is hereby incorporatedby reference in its entirety). Corso found six BCFA in the milk of 40women in southern Italy (Corso et al., “Gaschromatography-massSpectrometry Analysis of Fatty Acids in Human Milk from Forty PuerperaeLiving in Southern Italy,” Riv Eur Sci Med Farmacol 17:215-219 (1995),which is hereby incorporated by reference in its entirety), withanteiso-17:0 ranging from 0.12 to 0.93% of total fatty acids. Thisvariability is not unlike that for DHA; breastmilk DHA ranges between0.06% w/w and 1.40% w/w (Brenna et al., “Docosahexaenoic and ArachidonicAcid Concentrations in Human Breast Milk Worldwide,” Am J Clin Nutr85:1457-1464 (2007), which is hereby incorporated by reference in itsentirety). Many, but not all, breastmilk fatty acid concentrations areclosely linked to the dietary intake of the fatty acid or its precursor,including DHA to which the wide reported range is ascribed (Brenna etal., “Docosahexaenoic and Arachidonic Acid Concentrations in HumanBreast Milk Worldwide,” Am J Clin Nutr 85:1457-1464 (2007), which ishereby incorporated by reference in its entirety). It is most likelythat the absence of BCFA identification in most breast milk FA papers isdue to their low concentration, their appearance in a GC trace amidstthe major saturates and monounsaturates in the chromatogram, and thehistorical absence of a compelling metabolic role for them.

The exposure of the gut to BCFA in utero, and possibly from breastmilk,is greater than any other period of life, because BCFA are at tracelevels in normal foods. The unique niche represented by BCFA and othercomponents in the human gut may be important for establishing commensalbacteria during colonization. BCFA are prominent membrane components ofmany bacterial species (Kaneda et al., “Iso- and Anteiso-Fatty Acids inBacteria: Biosynthesis, Function, and Taxonomic Significance,” MicrobiolRev 55:288-302 (1991) and Huang et al., “Basic Characteristics ofSporolactobacillus inulinus BCRC 14647 for Potential ProbioticProperties,” Curr Microbiol 54:396-404 (2007), which are herebyincorporated by reference in their entirety). For instance, BCFAconstitute 95% of the FA in several bacilli and lactobacilli, includingSporolactobacillus inulinus, which has very recently been shown to be aprobiotic candidate (Huang et al., “Basic Characteristics ofSporolactobacillus inulinus BCRC 14647 for Potential ProbioticProperties,” Curr Microbiol 54:396-404 (2007), which is herebyincorporated by reference in its entirety). The FA of nineBifidobacterium strains include BCFA such as iso-14:0, anteiso-15:0,iso-16:0 and iso-18:0 at various levels (0.6-24.6% w/w). iso-14:0 is thesecond most abundant FA in Bifidobacterium breve with levels as high as24.6% w/w (Veerkamp et al., “Fatty Acid Composition of Bifidobacteriumand Lactobacillus Strains,” J Bacterial 108:861-867 (1971), which ishereby incorporated by reference in its entirety). It is reasonable tohypothesize that the presence of BCFA in the neonatal gut would alterthe mix of dominant species, favoring those organisms that use BCFA intheir membranes, and it is postulated that BCFA are a unique feature ofthe human fetal gut favoring the growth of commensal bacteria duringcolonization.

This hypothesis has implications for colonization of the GI tract ofvery premature infants, and may be a factor in the development ofnecrotizing enterocolitis (NEC), the etiology and pathogenesis of whichis not well understood (Caplan et al., “Bifidobacterial SupplementationReduces the Incidence of Necrotizing Enterocolitis in a Neonatal ratModel,” Gastroenterology 117:577-583 (1999) and Claud et al.,“Hypothesis: Inappropriate Colonization of the Premature Intestine CanCause Neonatal Necrotizing Enterocolitis,” Faseb J 15:1398-1403 (2001),which are hereby incorporated by reference in their entirety). NEC isone of the major causes of morbidity in premature infants (Caplan etal., “Bifidobacterial Supplementation Reduces the Incidence ofNecrotizing Enterocolitis in a Neonatal rat Model,” Gastroenterology117:577-583 (1999), which is hereby incorporated by reference in itsentirety) though it is certainly related to pathogen overgrowth(Hallstrom et al., “Effects of Mode of Delivery and NecrotisingEnterocolitis on the Intestinal Microflora in Preterm Infants,” Eur JClin Microbial Infect Dis 23:463-470 (2004), which is herebyincorporated by reference in its entirety). Leading hypotheses withempirical support are that NEC is related to prematurity, enteralfeeding, and bacterial colonization (Claud et al., “Hypothesis:Inappropriate Colonization of the Premature Intestine Can Cause NeonatalNecrotizing Enterocolitis,” Faseb J 15:1398-1403 (2001), which is herebyincorporated by reference in its entirety). Importantly, it has not beenobserved prenatally. NEC risk is higher among lower gestational ageinfants and is rare in term infants (Beeby et al., “Risk Factors forNecrotising Enterocolitis: the Influence of Gestational Age,” Arch DisChild 67:432-435 (1992), which is hereby incorporated by reference inits entirety). Breast milk consumption is associated with a lowerincidence of NEC (Claud et al., “Hypothesis: Inappropriate Colonizationof the Premature Intestine Can Cause Neonatal NecrotizingEnterocolitis,” Faseb J 15:1398-1403 (2001) and Lucas et al., “BreastMilk and Neonatal Necrotising Enterocolitis,” Lancet 336:1519-1523(1990), which are hereby incorporated by reference in their entirety).Although no specific pathogenic bacteria has been associated with NEC(Lucas et al., “Breast Milk and Neonatal Necrotising Enterocolitis,”Lancet 336:1519-1523 (1990), which is hereby incorporated by referencein its entirety), supplementation of premature animals and infants withprobiotic strains appear to reduce its incidence (Caplan et at.,“Bifidobacterial Supplementation Reduces the Incidence of NecrotizingEnterocolitis in a Neonatal rat Model,” Gastroenterology 117:577-583(1999) and Hoyos et al., “Reduced Incidence of Necrotizing EnterocolitisAssociated with Enteral Administration of Lactobacillus acidophilus andBifidobacterium infantis to Neonates in an Intensive Care Unit,” Int JInfect Dis 3:197-202 (1999), which are hereby incorporated by referencein their entirety). With these considerations, it is hypothesized thatBCFA have a role in enhancing proper GI colonization: vernix begins toappear around week 24 of gestation and accumulates as particulates inamniotic fluid toward term (Narendran et al., “Interaction betweenPulmonary Surfactant and Vernix: A Potential Mechanism for Induction ofAmniotic Fluid Turbidity,” Pediatr Res 48:120-124 (2000), which ishereby incorporated by reference in its entirety), thus, the GI tract ofvery premature infants is not exposed to vernix BCFA prenatally.Postnatally they would be exposed to BCFA if breastfed, but formula-fedpreterms would not be exposed to BCFA since they are not a component ofpreterm formulas. Finally, it is noted that the incidence of NEC dropsas gestational age approaches normal term. Therefore later termpremature infants would be exposed to some BCFA and may benefit if thehypothesis is correct.

The mass of BCFA entering and exiting the alimentary canal can beestimated. At term, amniotic fluid lipids are about 154 mg/L (Biezenskiet al., “Studies on the Origin of Amniotic Fluid Lipids I. NormalComposition,” Am J Obstet Gynecol 102:853-861 (1968), which is herebyincorporated by reference in its entirety), of which about 52 mg/L arephospholipids that are likely to originate as BCFA-free lung surfactant(Narendran et al., “Interaction between Pulmonary Surfactant and Vernix:A Potential Mechanism for Induction of Amniotic Fluid Turbidity,”Pediatr Res 48:120-124 (2000) and Rissmann et al., “New Insights intoUltrastructure, Lipid Composition and Organization of Vernix Caseosa,” JInvest Dermatol 126:1823-1833 (2006), which are hereby incorporated byreference in their entirety). Thus, the amniotic fluid vernix FAconcentration is about 102 mg/L. Of this, the measurements indicate that57% are FA, to yield 58 mg/L. The data (Table 2) further indicate that29% are BCFA, to yield 17 mg/L BCFA. The fetus is estimated to swallow200 to 500 ml/day of amniotic fluid near term (Pritchard et al.,“Deglutition by Normal and Anencephalic Fetuses,” Obstet Gynecol25:289-297 (1965) and Pritchard et al., “Fetal Swallowing and AmnioticFluid Volume,” Obstet Gynecol 28:606-610 (1966), which are herebyincorporated by reference in their entirety), and taking the midpoint ofthis range, 350 ml/day, 6 mg BCFA per day enter the fetal GI tractamounting to 30×6=180 mg BCFA in the last month of gestation. Meconiumis the output of the GI tract integrated from about 12 weeks gestation.Total meconium for 27 term infants was reported (Friel et al., “TraceElements in Meconium from Preterm and Full-term Infants,” Biol Neonate55:214-217 (1989), which is hereby incorporated by reference in itsentirety), to be 8.9 g wet weight, averaging 32% dry weight, or 2.8 g.The data indicate that about 0.55% is BCFA, or about 16 mg average totalBCFA in meconium. This value is an order of magnitude lower than theestimate of the BCFA swallowed in the last month of gestation, andsuggests that most of the BCFA disappear during transit. Thedistribution and structural characteristics of BCFA that do appear inmeconium reflect processing of vernix by the enterocytes. The presentinvention shows that C11-15 BCFA, as well as nearly all BCFA apart fromiso-BCFA, are absent from meconium and thus must have been metabolized.The nature of this metabolism remains to be determined, in part becauseBCFA and their interaction with human enterocytes has not been studied.

Chain elongation is one likely metabolic transformation that wouldexplain the absence of C11-15 BCFA, and preponderance of longer chainBCFA, in meconium. Suggestive evidence in support of this hypothesis isfound in the data of FIG. 1. The significantly greater level of meconiumiso-16:0 compared to vernix iso-16:0, is roughly the sum of vernixiso-14:0 and iso-16:0, consistent with the hypothesis that elongation ofvernix iso-14:0 adds to the existing iso-16:0. Similar observationsapply to meconium iso-20:0 and vernix iso-18:0 and iso-20:0.

Medium chain fatty acids (C8-C14) are commonly fed to premature infants,because they are efficiently absorbed through the gastric mucosa,directly transported to the liver via the portal vein, and oxidized bythe immature GI tract. Although the BCFA with 15 or fewer carbons areabsent from meconium, FIG. 4 shows that the FA n-14:0 and n-15:0 arepartially excreted. This observation implies that there is selectiveuptake and retention of BCFA by the fetal GI tract that may not operateas efficiently for the n-FA.

The present measurements of BCFA are in line with previous data. BCFAconstituted almost one third of all FA in vernix (Kaerkkaeinen et al.,“Lipids of Vernix Caseosa,” J Invest Dermatol 44:333-338 (1965) andNicolaides et al., “The Structures of the Branched Fatty Acids in theWax Esters of Vernix Caseosa Lipid,” Lipids 11:781-790 (1976), which arehereby incorporated by reference in their entirety), and the levels ofvernix SFA, MUFA and PUFA were within the range encompassed by previousreports (Rissmann et al., “New Insights into Ultrastructure, LipidComposition and Organization of Vernix Caseosa,” J Invest Dermatol126:1823-1833 (2006); Nicolaides et al., “Further Studies of theSaturated Methyl Branched Fatty Acids of Vernix Caseosa Lipid,” Lipids11:781-790 (1976); and Nicolaides et al., “The Structures of theBranched Fatty Acids in the Wax Esters of Vernix Caseosa Lipid,” Lipids11: 901-905 (1971), which are hereby incorporated by reference in theirentirety). Only odd numbered carbon anteiso BCFA were found, consistentwith some previous reports (Nicolaides et al., “Further Studies of theSaturated Methyl Branched Fatty Acids of Vernix Caseosa Lipid,” Lipids11:781-90 (1976); Nicolaides et al., “The Structures of the BranchedFatty Acids in the Wax Esters of Vernix Caseosa,” Lipids 6:901-905(1971) and Haahti et al., “Fatty Acids of Vernix Caseosa,” Scand J ClinLab Invest 13:70-73 (1961), which are hereby incorporated by referencein their entirety), but not with others (Rissmann et al., “New Insightsinto Ultrastructure, Lipid Composition and Organization of VernixCaseosa,” J Invest Dermatol 126:1823-1833 (2006) and Kaerkkaeinen etal., “Lipids of Vernix Caseosa,” J Invest Dermatol 44:333-338 (1965),which are hereby incorporated by reference in their entirety). BCFAaveraged 17% w/w of all FA in meconium in the samples. The singleprevious study showing BCFA in meconium reported only on the free fattyacid fraction and used GC with retention time matching foridentification. iso FA with 22 and 24 carbons were identified at 4% w/wand 6% w/w respectively, and nine other iso-BCFA were tentativelyassigned (C14-21, 25) with no percent fraction provided.

Though five anteiso BCFA were detected in vernix, anteiso-17:0 was thesole anteiso BCFA detected in meconium, and there is no obviousexplanation as to why this was the case. Weanling rats fed 100 mg/weekanteiso-17:0 in an otherwise fat free diet excreted 8-10% in the fecesand stored a similar amount in adipose tissue (Livingston et al., “TheMetabolism in the Rat of Naturally Occurring (+)-14-MethylhexadecanoicAcid,” Biochem J 65:438-440 (1957) which is hereby incorporated byreference in its entirety), and apparently also converted a small amountanteiso-15:0. The remaining 80% was metabolized to substances other thananteiso FA. The levels of anteiso-17:0 have been reported to be thehighest among all BCFA in at least one study of breastmilk (Gibson etal., “Fatty Acid Composition of Human Colostrum and Mature Breast Milk,”Am J Clin Nutr 34:252-257 (1981), which is hereby incorporated byreference in its entirety), and it is notable that anteiso-17:0 is amajor lipids constituent of many bacterial membrane (Kaneda et al.,“Iso- and Anteiso-Fatty Acids in Bacteria: Biosynthesis, Function, andTaxonomic Significance,” Microbiol Rev 55:288-302 (1991), which ishereby incorporated by reference in its entirety).

The combined levels of the middle chain monomethyl and dimethyl BCFA inthe vernix samples were similar to the levels reported in a singlevernix sample by Nicolaides & Apon (Nicolaides et al., “The Structuresof the Branched Fatty Acids in the Wax Esters of Vernix Caseosa Lipid,”Lipids 11:781-790 (1976), which is hereby incorporated by reference inits entirety). In the sample of 18 newborns, the average proportions ofdimethyl monomethyl BCFA dominated over middle chain monomethyl BCFA.The first methyl branch in the dimethyl BCFA was located predominantlyon the fourth carbon of the chain, consistent with previous findings(Nicolaides et al., “The Structures of the Branched Fatty Acids in theWax Esters of Vernix Caseosa Lipid,” Lipids 11:781-790 (1976) andNicolaides et al., “The Structures of the Branched Fatty Acids in theWax Esters of Vernix Caseosa,” Lipids 6:901-905 (1971), which are herebyincorporated by reference in its entirety). However, in the presentinvention, the second methyl branch in half of the dimethyl BCFA waslocated on an odd numbered carbon, and in almost all the dimethyl BCFA,this methyl branch was located on the anteiso carbon of the FA chain.

In summary, there are dramatic and systematic differences in BCFAcomposition between vernix and meconium, indicating that BCFA areactively metabolized in the fetal GI tract. This observation impliesthat vernix should be considered a nutritional agent, and that BCFA area normal and quantitatively substantial component of the normal termnewborn GI tract. Further studies are warranted to understand the uptakeand metabolism of BCFA by enterocytes, and the role of BCFA duringbacterial colonization. The absence of vernix, and BCFA, in the GI tractof very premature, formula-fed infants may have a role in the etiologyof NEC, among the most devastating conditions facing the preterm infant.

Materials and Methods for Examples 6-7 Caco-2 Cells

Caco-2 cells are a human colon cancer cell line which was introduced inthe 1980s as a model for enterocyte metabolism and rapidly became astandard in vitro model for studies of absorption and metabolism inenterocytes. The cells are often cultured on microporous membranes wherethey assume a polarized configuration, with villi developing on theapical side and secretion of chylomicrons and very low densitylipoproteins on the basolateral side (Traber et al., “PolarizedSecretion of Newly Synthesized Lipoproteins by the Caco-2 HumanIntestinal Cell Line,” J Lipid Res 28:1350-63 (1987) and Luchoomun etal., “Assembly and Secretion of Chylomicrons by Differentiated Caco-2Cells. Nascent Triglycerides and Preformed Phospholipids arePreferentially Used for Lipoprotein Assembly,” J Biol Chem 274:19565-72(1999), which are hereby incorporated by reference in their entirety).Among the limitations of in vitro systems is their metabolic similarityto the in vivo condition. Caco-2 cells differentiate and make microvilliresembling those of the upper intestine, however they are not as denseas the in vivo condition, and are considered similar to fetal cells(Blais et al., “Common Characteristics for Na+-dependent Sugar Transportin Caco-2 Cells and Human Fetal Colon,” J Membr Biol 99:113-25 (1987)and Zweibaum et al., “Sucrase-isomaltase: A Marker of Foetal andMalignant Epithelial Cells of the Human Colon,” Int J Cancer 32:407-12(1983), which are hereby incorporated by reference in their entirety).Moreover, the normal contents of the fetal and neonatal GI tract aremuch less varied than the contents of the adult GI tract. The fetal gutprocesses amniotic fluid while the neonatal GI tract processes breastmilk or its substitutes. The adult must process any conceivable food, acomplication that requires careful design of simulated digestive modelsystems prior to study of food absorption by Caco-2 (Fairweather-Tait etal., “The Usefulness of In Vitro Models to Predict the Bioavailabilityof Iron and Zinc: A Consensus Statement from the HarvestPlus ExpertConsultation,” Int J Vitam Nutr Res 75:371-4 (2005), which is herebyincorporated by reference in its entirety).

These considerations suggest that Caco-2 cells are most appropriate formodeling the present invention. Apart from limitations in extrapolatingresults of Caco-2 data to the in vivo situation, experts consideringBCFA as nutritional or therapeutic agent will wish to have data onabsorption and metabolism in Caco-2 cells as reference data, therebyjustifying the use of the model.

Caco-2 cells were grown to confluence on a solid bottom, multi-wellplates in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% v:vfetal bovine serum. Twenty-four hours prior to treatment with BCFA, DMEMwas replaced with MEM (Minimum Essential Medium). Two wells wereincubated with 1.2 mmol each of a mixture of four, pure BCFA (iso-14:0,anteiso-17:0, iso-18:0, iso-20:0) as free fatty acids (FFA) obtainedfrom Larodan Lipids (Malmo, Sweden). Two others served as controls.Cells were incubated for 18 hours, after which media was removed and thecells were thoroughly and carefully washed three times. Cell lipids wereextracted and FAME prepared for GC/MS.

Example 6 BCFA Interaction With Caco-2 Cells: Caco-2 Cells Readily TakeUp BCFA From Media and Biosynthesize New BCFA From Exogenous BCFA

FIG. 5 outlines the GC/MS data, presented as reconstructed ionchromatograms (RIC) of the section of the chromatogram corresponding tothe elution time of BCFA. In FIG. 5A, the top panel shows the controlcells C14-C18 saturated and monounsaturated FA. There is no evidence ofBCFA above 0.1% of fatty acids in these data. In FIG. 5B, the middlepanel is a RIC of the BCFA mixture used to treat the cells. In FIG. 5C,the bottom panel is the RIC of the Caco-2 cells incubated for 18 hourswith BCFA. Careful inspection of the three panels reveals that the RICof panel (FIG. 5C) appears very much like a composite of thechromatograms in (FIG. 5A) and (FIG. 5B). The cells have taken up alarge amount of BCFA, such that they are now the dominant saturated FA.However, a peak appearing in the treated cells but neither the controlcells (FIG. 5A) nor the BCFA mixture (FIG. 5B) is iso-16:0. This BCFAcan only have arisen by biosynthesis, likely to be by elongation ofiso-14:0. It was calculated that the newly biosynthesized iso-16:0 isabout 10% of the iso-14:0 peak, indicating substantial conversionactivity over 18 h if all iso-16:0 arises from chain elongation ofiso-14:0. In these cells, BCFA constitute 33% of the total FA, and 53%of the saturated FA. This contrasts with 1.7% 20:4 (arachidonic acid)and 0.86% docosahexaenoic acid (DHA), two LCPUFA of considerablemetabolic importance, in these cells.

FIGS. 6 and 7 present profiles of BCFA within phospholipids (PL) andmonoacylglycerols (MAG) of the same BCFA-treated cells shown in FIG. 5.FIG. 6 shows that BCFA are incorporated into PL at about half the levelof endogenous FA (compare heights of, e.g., iso-14:0 vs n-16:0 in FIG.6B). Note also that the iso-20:0 peak is much smaller compared to otherBCFA (compare FIGS. 5A, 5C, 6B, and 7). A selected ion chromatogram andmass spectra are also shown to further illustrate positiveidentification of the 16:0 isomers as an example.

FIG. 7 shows that BCFA are by far the dominant FA in MAG (compareiso-14:0 vs n-16:0). Newly synthesized iso-16:0 is very low compared toiso-14:0 in this fraction. It is likely that free BCFA entering thecells are initially incorporated into MAG and thus the concentration ishighest, and relative appearance of newly synthesized iso-16:0 is low.

Example 7 Caco-2 Cells Selectively Incorporate BCFA Into Lipid ClassesBased on Chain Length and on Lipid Class

FIG. 8 is a summary of the BCFA profiles in each sample, showing thatthe initial FFA mix was transformed into PL and MAG, with chain lengthselectivities that could help explain the vernix/meconium BCFA profiles.

A mixture of BCFA was used to generate this preliminary data so toprovide a basic simulation of vernix BCFA without protein or otherconfounding compounds. Cells are treated with individual BCFA,anteiso-17:0, iso-18:0, and others, to establish whether iso-14:0 iselongated or iso-18:0 is chain shortened to yield iso-16:0, forinstance.

The data in the present invention is consistent with the hypothesis'sprediction that the difference in distribution between vernix andmeconium BCFA arises, at least in part, due to uptake and differentialmetabolism of BCFA. These are among the rare data on BCFA metabolism inmammals apart from studies in skin and surface glands.

BCFA are a major component of the normal term infant alimentary canal.The present invention will develop the knowledge on BCFA metabolism inintestinal cells, focused on the practical issue of NEC, which developsin immature infants with negligible or low gut BCFA (simulated bycontrol cells).

Example 8 Anaerobic Cultures and Vernix

Anaerobic microbial growth studies were performed, and whether vernixinfluences proliferation of commensal or pathogenic bacteria wasexamined. Two commensal bacteria (Bifidobacteria breve and S. inulinus)and three pathogens (C perfringens, C difficile, both obligateanaerobes, and E coli, a facultative anaerobe) were grown anaerobicallyin pure culture. Two nutrient media were used (Trypticase Soy Broth andNutrient Broth (“NB”, Difco)). Vernix leftover from that used togenerate data above was dissolved in ethanol, autoclaved, and added tocultures (Vernix). Ethanol alone was used as a control (C).

FIG. 9 shows that B. breve vernix developed optical density 80% fasterthan the controls. The growth enhancement in S. inulinus was dramatic inboth media. In contrast, the pathogens in the right panels are notdifferent between vernix and the controls.

The present invention provides preliminary evidence that vernix itselfinfluences gut flora. Upon reflection, it makes sense that friendlybacterial growth is enhanced by vernix, the normal contents of the termnewborn human gut, and that vernix should put opportunistic pathogens atno special advantage.

It cannot be confirmed from the present invention that it was the BCFAcomponent of vernix that enhanced growth. However, it is likely that thebioactivity of vernix peptides are substantially impaired or inactivatedafter dissolution in ethanol and autoclaving, while the activities ofsaturated lipids are not affected. Second, the magnitude of growth byeach bacteria is not an indicator of its competitive fitness; one cannotconclude that C. perfringens is the most fit organism because of itsfast growth characteristics in this medium. These data apply only to theinfluence of sterilized vernix per se on growth. Properly controlledcompetitive growth assays are required for this purpose. It is importantto note that these are all the data from the first attempt with thesecultures. The conditions have not yet been optimized. For instance, anew paper shows that bile enhances uptake of BCFA by the bifidobacteria(Ruiz et al., “Cell Envelope Changes in Bifidobacterium animalis ssp.lactis as a Response to Bile,” FEMS Microbiol Lett 274:316-22 (2007),which is hereby incorporated by reference in its entirety), andcontinuing studies will take this into account.

The dose of BCFA was 23 μg per 20 ml culture, or about 1 μg/ml, based onaddition of 380 μg vernix (dry weight). This can be compared to theinfant's estimated exposure. Vernix particles that are relatively dilutein gulped amniotic fluid will be rapidly concentrated in the newborn gutas water and electrolytes are absorbed. Amniotic fluid contains 138 μglipids/ml between 34-40 weeks gestation, and >39 weeks it contains 386μg lipids/ml (Lentner C., Geigy Scientific Tables, 8th ed. New Jersey:Ciba-Geigy, (1981), which is hereby incorporated by reference in itsentirety), most of which are vernix, with surfactant and other minorlipids present. This concentration rises dramatically upon absorption ofwater and conversion of the liquid amniotic fluid into a paste in theupper infant gut. Based on the measurements of the concentration of BCFAin vernix (6% dry weight) and meconium (0.5% dry wt), a totalconcentration of 1 mg BCFA/ml meconium can be estimated. Based on thisanalysis, microorganisms arriving in the gut after birth will thereforeencounter much higher concentrations of BCFA, though it cannot be saidif they will behave similarly to BCFA dissolved in ethanol.Nevertheless, the present invention shows that low levels of vernixcomponents, compared to the gut environment, may exert major effects.

It is noted that the newest (preliminary) data are consistent with thehypothesis that vernix enhances the growth of commensal organisms,leaving opportunistic pathogen growth unaffected. There is evidence,however, that vernix lipids per se have antimicrobial properties, andinteract synergistically with an antimicrobial vernix peptide to inhibitgrowth of at least one model organism. Vernix lipids inhibited thegrowth of Bacillus megaterium compared to a no lipid control. Moreover,the addition of vernix peptide LL-37, when mixed in a ratio of 3:1 withvernix lipids, results in further growth inhibition (Tollin et al.,“Vernix Caseosa as a Multi-component Defence System Based onPolypeptides, Lipids and Their Interactions,” Cell Mol Life Sci62:2390-9 (2005), which is hereby incorporated by reference in itsentirety). Importantly, that study detected 23% “unidentified fattyacids” among their vernix fatty acids, which can safely be assumed to beoverwhelmingly BCFA.

Importantly, the proposed studies, including competition experiments,naturally evaluate the relative pro-commensal, anti-pathogen(“antimicrobial”) properties of vernix and its lipids when focused uponthe hypothesis, as cast.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of preventing or treating a gastrointestinal condition in asubject, said method comprising: administering one or more branchedchain fatty acid to the subject under conditions effective to prevent ortreat the gastrointestinal condition in the subject.
 2. The method ofclaim 1 further comprising: selecting a subject in need of treatment ofthe gastrointestinal condition prior to said administering.
 3. Themethod of claim 1 further comprising: selecting a subject in need ofprevention of the gastrointestinal condition prior to saidadministering.
 4. The method of claim 1, wherein the gastrointestinalcondition is mediated by infection of the subject's gastrointestinaltract by a pathogenic bacteria.
 5. The method of claim 4, wherein thegastrointestinal condition is necrotizing enterocolitis.
 6. The methodof claim 1, wherein the gastrointestinal condition requires microbialcolonization of the subject's gastrointestinal tract.
 7. The method ofclaim 6, wherein colonization of the subject's gastrointestinal tract iscarried out in conjunction with an antibiotic treatment.
 8. The methodof claim 1, wherein the gastrointestinal condition is a disease of theintestine involving inflammation.
 9. The method of claim 8, wherein thegastrointestinal condition is inflammatory bowel disease.
 10. The methodof claim 1, wherein the one or more branched chain fatty acid is a C₁₁to C₂₆ branched chain fatty acid or mixtures thereof.
 11. The method ofclaim 1, wherein the one or more branched chain fatty acid is selectedfrom the group consisting of 4,7-dimethyl-nonanoic acid,4,8-dimethyl-decanoic acid, 8-methyl-undecanoic acid, iso-dodecanoicacid, 4,8-dimethyl-undecanoic acid, 4,9-dimethyl-undecanoic acid,iso-tridecanoic acid, anteiso-tridecanoic acid, 4,10-dimethyl-dodecanoicacid, iso-tetradecanoic acid, 4,11-dimethyl-tridecanoic acid,iso-pentadecanoic acid, anteiso-pentadecanoic acid,8,10-dimethyl-tetradecanoic acid, 4,12-dimethyl-tetradecanoic acid,iso-hexadecanoic acid, 2-methyl hexadecanoic acid,4,11-dimethyl-pentadecanoic acid, 4,13-dimethyl-pentadecanoic acid,iso-heptadecanoic acid, anteiso heptadecanoic acid, iso-octadecanoicacid, iso-eicosanoic acid, anteiso-heneicosanoic acid, iso-dodecanoicacid, iso-tetracosanoic acid, iso-pentacosanoic acid,anteiso-pentacosanoic acid, iso-hexacosanoic acid, phytanic acid,pristanic acid, and mixtures thereof.
 12. The method of claim 11,wherein the one or more branched chain fatty acid is selected from thegroup consisting of iso-hexadecanoic acid, anteiso-hexadecanoic acid,4,13-dimethylpentadecanoic acid, phytanic acid, pristanic acid, andmixtures thereof.
 13. The method of claim 1, wherein the one or morebranched chain fatty acid is selected from the group consisting ofsaturated and monounsaturated fatty acids and mixtures thereof.
 14. Themethod of claim 13, wherein the one or more branched chain fatty acid isa branched form of a fatty acid selected from the group consisting of anoctanoic acid, a decanoic acid, a lauric acid, a myristic acid, apalmitic acid, a stearic acid, an eicosanoic acid, a palmitoliec acid,an oleic acid, and mixtures thereof.
 15. The method of claim 1, whereinthe subject is human.
 16. The method of claim 15, wherein the subject isa fetus.
 17. The method of claim 15, wherein the subject is an infant.18. The method of claim 17, wherein the subject is a newborn infant. 19.The method of claim 15, wherein the subject is a child.
 20. The methodof claim 15, wherein the subject is an adult.
 21. A method of promotinggastrointestinal health in a subject, said method comprising:administering one or more branched chain fatty acid to the subject underconditions effective to promote gastrointestinal health in the subject.22. The method of claim 21 further comprising: selecting a subject inneed of promoting gastrointestinal health prior to said administering.23. The method of claim 21, wherein the one or more branched chain fattyacid is a C₁₁ to C₂₆ branched chain fatty acid or mixtures thereof. 24.The method of claim 21, wherein the one or more branched chain fattyacid is selected from the group consisting of 4,7-dimethyl-nonanoicacid, 4,8-dimethyl-decanoic acid, 8-methyl-undecanoic acid,iso-dodecanoic acid, 4,8-dimethyl-undecanoic acid,4,9-dimethyl-undecanoic acid, iso-tridecanoic acid, anteiso-tridecanoicacid, 4,10-dimethyl-dodecanoic acid, iso-tetradecanoic acid,4,11-dimethyl-tridecanoic acid, iso-pentadecanoic acid,anteiso-pentadecanoic acid, 8,10-dimethyl-tetradecanoic acid,4,12-dimethyl-tetradecanoic acid, iso-hexadecanoic acid, 2-methylhexadecanoic acid, 4,11-dimethyl-pentadecanoic acid,4,13-dimethyl-pentadecanoic acid, iso-heptadecanoic acid, anteisoheptadecanoic acid, iso-octadecanoic acid, iso-eicosanoic acid,anteiso-heneicosanoic acid, iso-dodecanoic acid, iso-tetracosanoic acid,iso-pentacosanoic acid, anteiso-pentacosanoic acid, iso-hexacosanoicacid, phytanic acid, pristanic acid, and mixtures thereof.
 25. Themethod of claim 24, wherein the one or more branched chain fatty acid isselected from the group consisting of iso-hexadecanoic acid,anteiso-hexadecanoic acid, 4,13-dimethylpentadecanoic acid, phytanicacid, pristanic acid, and mixtures thereof.
 26. The method of claim 21,wherein the one or more branched chain fatty acid is selected from thegroup consisting of saturated and monounsaturated fatty acids andmixtures thereof.
 27. The method of claim 26, wherein the one or morebranched chain fatty acid is a branched form of a fatty acid selectedfrom the group consisting of an octanoic acid, a decanoic acid, a lauricacid, a myristic acid, a palmitic acid, a stearic acid, an eicosanoicacid, a palmitoliec acid, an oleic acid, and mixtures thereof.
 28. Themethod of claim 21, wherein the subject is human.
 29. A method ofpromoting propagation of probiotic organisms, said method comprising:providing a population of cells comprising probiotic organisms andadministering one or more branched chain fatty acid to the population ofcells under conditions effective to promote propagation of probioticorganisms in the population of cells.
 30. The method of claim 29,wherein the one or more branched chain fatty acid is a C₁₁ to C₂₆branched chain fatty acid or mixtures thereof.
 31. The method of claim29, wherein the one or more branched chain fatty acid is selected fromthe group consisting of 4,7-dimethyl-nonanoic acid,4,8-dimethyl-decanoic acid, 8-methyl-undecanoic acid, iso-dodecanoicacid, 4,8-dimethyl-undecanoic acid, 4,9-dimethyl-undecanoic acid,iso-tridecanoic acid, anteiso-tridecanoic acid, 4,10-dimethyl-dodecanoicacid, iso-tetradecanoic acid, 4,11-dimethyl-tridecanoic acid,iso-pentadecanoic acid, anteiso-pentadecanoic acid,8,10-dimethyl-tetradecanoic acid, 4,12-dimethyl-tetradecanoic acid,iso-hexadecanoic acid, 2-methyl hexadecanoic acid,4,11-dimethyl-pentadecanoic acid, 4,13-dimethyl-pentadecanoic acid,iso-heptadecanoic acid, anteiso heptadecanoic acid, iso-octadecanoicacid, iso-eicosanoic acid, anteiso-heneicosanoic acid, iso-dodecanoicacid, iso-tetracosanoic acid, iso-pentacosanoic acid,anteiso-pentacosanoic acid, iso-hexacosanoic acid, phytanic acid,pristanic acid, and mixtures thereof.
 32. The method of claim 31,wherein the one or more branched chain fatty acid is selected from thegroup consisting of iso-hexadecanoic acid, anteiso-hexadecanoic acid,4,13-dimethylpentadecanoic acid, phytanic acid, pristanic acid, andmixtures thereof.
 33. The method of claim 29, wherein the one or morebranched chain fatty acid is selected from the group consisting ofsaturated and monounsaturated fatty acids and mixtures thereof.
 34. Themethod of claim 33, wherein the one or more branched chain fatty acid isa branched form of a fatty acid selected from the group consisting of anoctanoic acid, a decanoic acid, a lauric acid, a myristic acid, apalmitic acid, a stearic acid, an eicosanoic acid, a palmitoliec acid,an oleic acid, and mixtures thereof.
 35. The method of claim 29, whereinthe method is carried out in vitro.
 36. The method of claim 29, whereinthe method is carried out in vivo.
 37. The method of claim 36 furthercomprising: selecting a subject in need of probiotic organismpropagation prior to said administering.
 38. The method of claim 29,wherein the probiotic organism comprises an organism selected from thegroup consisting of Lactobacillus species, Bifidobacterium species,other lactic acid bacteria, nonlactic acid bacteria, and mixturesthereof.
 39. The method of claim 38, wherein the probiotic organismcomprises a Lactobacillus species selected from the group consisting ofL. acidophilus, L. amylovorus, L. brevis, L. casei, L. casei subsp.rhamnosus (Lactobacillus GG), L. caucasicus, L. crispatus, L.delbrueckii subsp. bulgaricus (L. bulgaricus), L. fermentum (L.fermenti), L. gasseri, L. helveticus, L. johnsonii, L. lactis, L.leichmannii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, andmixtures thereof.
 40. The method of claim 38, wherein the probioticorganism comprises a Bifidobacterium species selected from the groupconsisting of B. adolescentis, B. bifidum, B. breve, B. infantis, B.lactis (B. animalis), B. licheniformis, B. longum, and mixtures thereof.41. The method of claim 38, wherein the probiotic organism comprises alactic acid bacteria selected from the group consisting of Enterococcusfaecium, Lactococcus lactis, Leuconstoc mesenteroides, Pediococcusacidilactici, Streptococcus thermophilus, and mixtures thereof.
 42. Themethod of claim 38, wherein the probiotic organism comprises a nonlacticacid bacteria selected from the group consisting of Bacillus subtilis,Escherichia coli strain nissle, Saccharomyces boulardii, Saccharomycescerevisiae, and mixtures thereof.
 43. A formulation comprising: one ormore branched chain fatty acids and an aqueous phase emulsified withsaid one or more branched chain fatty acids, wherein said formulationcomprises over 25 wt % of said one or more branched chain fatty acids.44. The formulation of claim 43, wherein the one or more branched chainfatty acid is a C₁₁ to C₂₆ branched chain fatty acid or a mixturethereof.
 45. The formulation of claim 43, wherein the one or morebranched chain fatty acid is selected from the group consisting of4,7-dimethyl-nonanoic acid, 4,8-dimethyl-decanoic acid,8-methyl-undecanoic acid, iso-dodecanoic acid, 4,8-dimethyl-undecanoicacid, 4,9-dimethyl-undecanoic acid, iso-tridecanoic acid,anteiso-tridecanoic acid, 4,10-dimethyl-dodecanoie acid,iso-tetradecanoic acid, 4,11-dimethyl-tridecanoic acid,iso-pentadecanoic acid, anteiso-pentadecanoic acid,8,10-dimethyl-tetradecanoic acid, 4,12-dimethyl-tetradecanoic acid,iso-hexadecanoic acid, 2-methyl hexadecanoic acid,4,11-dimethyl-pentadecanoic acid, 4,13-dimethyl-pentadecanoic acid,iso-heptadecanoic acid, anteiso heptadecanoic acid, iso-octadecanoicacid, iso-eicosanoic acid, anteiso-heneicosanoic acid, iso-dodecanoicacid, iso-tetracosanoic acid, iso-pentacosanoic acid,anteiso-pentacosanoic acid, iso-hexacosanoic acid, phytanic acid,pristanic acid, and mixtures thereof.
 46. The formulation of claim 45,wherein the one or more branched chain fatty acid is selected from thegroup consisting of iso-hexadecanoic acid, anteiso-hexadecanoic acid,4,13-dimethylpentadecanoic acid, phytanic acid, pristanic acid, andmixtures thereof.
 47. The formulation of claim 43, wherein the one ormore branched chain fatty acid is selected from the group consisting ofsaturated and monounsaturated fatty acids and mixtures thereof.
 48. Theformulation of claim 47, wherein the one or more branched chain fattyacid is a branched form of a fatty acid selected from the groupconsisting of an octanoic acid, a decanoic acid, a lauric acid, amyristic acid, a palmitic acid, a stearic acid, an eicosanoic acid, apalmitoliec acid, an oleic acid, and mixtures thereof.
 49. Theformulation of claim 45, wherein the formulation comprises up to 70 wt %of said one or more branched chain fatty acid.
 50. The formulation ofclaim 45, further comprising: an emulsifier.
 51. The formulation ofclaim 45, wherein said one or more branched chain fatty acid comprisesup to 20 wt % of isomyristic acid, isopentadecoanoic acid,anteisopentadecanoic acid, isopalmitic acid, isoeicosanoic acid,anteisoeicosanoic acid, isodocosanoic acid, isotetracosanoic acid,isohexacosanoic acid, or combinations thereof.